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Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process.

Treeck M, Zacherl S, Herrmann S, Cabrera A, Kono M, Struck NS, Engelberg K, Haase S, Frischknecht F, Miura K, Spielmann T, Gilberger TW - PLoS Pathog. (2009)

Bottom Line: We identify several residues in the cytoplasmic tail that are essential for AMA-1 function.We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes.We show that the cytoplasmic domain of AMA-1 is phosphorylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Parasitology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.

ABSTRACT
A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

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Functional analysis of the cytoplasmic domain of AMA-1.(A) Schematic representations of the mutations introduced into the cytoplasmic domain of W2mef-derived AMA-1. (B) Alignment of the cytoplasmic domain of various AMA-1 homologues. The conservation is scored and colour-coded by PRALINE (www.ibi.vu.nl). The scoring scheme works from 0 (for the least-conserved alignment position) up to 10 (for the most conserved position). Amino acids predicted by NetPhos (www.cbs.dtu.dk/services/NetPhos) to be phosphorylated in the plasmodial protein are scaled up according to their relative predicted probabilities. (C) Alignment of the cytoplasmic domain of AMA-1 with the cytoplasmic domain of EBA-175 and Rh2b. (D) Expression of the cytoplasmic domain mutants was verified by Western blot analysis using anti-GFP antibodies. (E) Invasion inhibition assays with all cytoplasmic domain mutants were performed in the presence of 100 µg/ml R1 peptide. Error bars correspond to standard deviation. All experiments were performed in triplicate in two independent experiments. The parasite line AMA-1W2mef-GFP served as a positive control. Except for the mutation of a conserved tyrosine (YD), all introduced mutations lead to a complete functional inactivation (in the case of the mutant DE, to a drastically reduced function) of AMA-1-GFP.
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ppat-1000322-g004: Functional analysis of the cytoplasmic domain of AMA-1.(A) Schematic representations of the mutations introduced into the cytoplasmic domain of W2mef-derived AMA-1. (B) Alignment of the cytoplasmic domain of various AMA-1 homologues. The conservation is scored and colour-coded by PRALINE (www.ibi.vu.nl). The scoring scheme works from 0 (for the least-conserved alignment position) up to 10 (for the most conserved position). Amino acids predicted by NetPhos (www.cbs.dtu.dk/services/NetPhos) to be phosphorylated in the plasmodial protein are scaled up according to their relative predicted probabilities. (C) Alignment of the cytoplasmic domain of AMA-1 with the cytoplasmic domain of EBA-175 and Rh2b. (D) Expression of the cytoplasmic domain mutants was verified by Western blot analysis using anti-GFP antibodies. (E) Invasion inhibition assays with all cytoplasmic domain mutants were performed in the presence of 100 µg/ml R1 peptide. Error bars correspond to standard deviation. All experiments were performed in triplicate in two independent experiments. The parasite line AMA-1W2mef-GFP served as a positive control. Except for the mutation of a conserved tyrosine (YD), all introduced mutations lead to a complete functional inactivation (in the case of the mutant DE, to a drastically reduced function) of AMA-1-GFP.

Mentions: The complementation assay provided the basis for analyzing the phenotypic effects of various deletions and mutations introduced into the AMA-1 protein and as such to functionally characterize different parts of the AMA-1 molecule. First, we analyzed the effect of the cytoplasmic domain deletion. In the presence of the R1 peptide, W2mef-derived AMA-1Δtail-GFP could not rescue the invasion capability, unlike the full-length W2mef AMA-1-GFP (Figure 4E). Thus, although trafficking of AMA-1 does not require the cytoplasmic domain, invasion inhibition by R1 clearly demonstrates that the cytoplasmic domain (AMA-1Δtail-GFP) plays an essential role in the invasion process. To further dissect the role of the cytoplasmic domain we introduced multiple alterations into this domain and analyzed their functional consequences.


Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process.

Treeck M, Zacherl S, Herrmann S, Cabrera A, Kono M, Struck NS, Engelberg K, Haase S, Frischknecht F, Miura K, Spielmann T, Gilberger TW - PLoS Pathog. (2009)

Functional analysis of the cytoplasmic domain of AMA-1.(A) Schematic representations of the mutations introduced into the cytoplasmic domain of W2mef-derived AMA-1. (B) Alignment of the cytoplasmic domain of various AMA-1 homologues. The conservation is scored and colour-coded by PRALINE (www.ibi.vu.nl). The scoring scheme works from 0 (for the least-conserved alignment position) up to 10 (for the most conserved position). Amino acids predicted by NetPhos (www.cbs.dtu.dk/services/NetPhos) to be phosphorylated in the plasmodial protein are scaled up according to their relative predicted probabilities. (C) Alignment of the cytoplasmic domain of AMA-1 with the cytoplasmic domain of EBA-175 and Rh2b. (D) Expression of the cytoplasmic domain mutants was verified by Western blot analysis using anti-GFP antibodies. (E) Invasion inhibition assays with all cytoplasmic domain mutants were performed in the presence of 100 µg/ml R1 peptide. Error bars correspond to standard deviation. All experiments were performed in triplicate in two independent experiments. The parasite line AMA-1W2mef-GFP served as a positive control. Except for the mutation of a conserved tyrosine (YD), all introduced mutations lead to a complete functional inactivation (in the case of the mutant DE, to a drastically reduced function) of AMA-1-GFP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654807&req=5

ppat-1000322-g004: Functional analysis of the cytoplasmic domain of AMA-1.(A) Schematic representations of the mutations introduced into the cytoplasmic domain of W2mef-derived AMA-1. (B) Alignment of the cytoplasmic domain of various AMA-1 homologues. The conservation is scored and colour-coded by PRALINE (www.ibi.vu.nl). The scoring scheme works from 0 (for the least-conserved alignment position) up to 10 (for the most conserved position). Amino acids predicted by NetPhos (www.cbs.dtu.dk/services/NetPhos) to be phosphorylated in the plasmodial protein are scaled up according to their relative predicted probabilities. (C) Alignment of the cytoplasmic domain of AMA-1 with the cytoplasmic domain of EBA-175 and Rh2b. (D) Expression of the cytoplasmic domain mutants was verified by Western blot analysis using anti-GFP antibodies. (E) Invasion inhibition assays with all cytoplasmic domain mutants were performed in the presence of 100 µg/ml R1 peptide. Error bars correspond to standard deviation. All experiments were performed in triplicate in two independent experiments. The parasite line AMA-1W2mef-GFP served as a positive control. Except for the mutation of a conserved tyrosine (YD), all introduced mutations lead to a complete functional inactivation (in the case of the mutant DE, to a drastically reduced function) of AMA-1-GFP.
Mentions: The complementation assay provided the basis for analyzing the phenotypic effects of various deletions and mutations introduced into the AMA-1 protein and as such to functionally characterize different parts of the AMA-1 molecule. First, we analyzed the effect of the cytoplasmic domain deletion. In the presence of the R1 peptide, W2mef-derived AMA-1Δtail-GFP could not rescue the invasion capability, unlike the full-length W2mef AMA-1-GFP (Figure 4E). Thus, although trafficking of AMA-1 does not require the cytoplasmic domain, invasion inhibition by R1 clearly demonstrates that the cytoplasmic domain (AMA-1Δtail-GFP) plays an essential role in the invasion process. To further dissect the role of the cytoplasmic domain we introduced multiple alterations into this domain and analyzed their functional consequences.

Bottom Line: We identify several residues in the cytoplasmic tail that are essential for AMA-1 function.We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes.We show that the cytoplasmic domain of AMA-1 is phosphorylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Parasitology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.

ABSTRACT
A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus